Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
  • C12N1/16—Yeasts; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
  • C12C11/00—Fermentation processes for beer
  • C12C11/003—Fermentation of beerwort
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
  • C12C12/00—Processes specially adapted for making special kinds of beer
  • C12C12/002—Processes specially adapted for making special kinds of beer using special microorganisms
  • C12C12/006—Yeasts
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
  • C12N1/145—Fungal isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
  • C12N1/16—Yeasts; Culture media therefor
  • C12N1/18—Baker's yeast; Brewer's yeast
  • C12N1/185—Saccharomyces isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
  • C12C2200/00—Special features
  • C12C2200/05—Use of genetically modified microorganisms in the preparation of beer
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi
  • C12R2001/85—Saccharomyces

Definitions

• the GY7B yeast strain was deposited, in accordance with the Budapest Treaty, with the American Type Culture Collection (ATCC®) on July 19, 2018, under Accession Number
• yeast strain categories examples include “baker's yeast” (which is a leavening agent) and “brewer's yeast” (which is used for alcoholic fermentation processes). It should be noted that, within each category, specific strains can produce distinct metabolic byproducts, which alter the properties of the food products in which they are incorporated.
• LAB may be added to wort between the mash and the boil, in a technique called kettle-souring. More fully developed sour flavors require the use of bacteria in the fermenter or during maturation of beer in bright tanks, barrels, or foeders, which can take 2 months to 2 years to fully develop.
• the invention provides a method of producing a yeast-fermented beverage.
• the invention further provides a yeast strain able to produce a yeast-fermented beverage with a low pH and a sour taste without the use of lactic acid producing bacteria.
• the invention further provides compositions comprising a yeast strain contemplated within the invention.
• the invention further provides a kit comprising a yeast strain contemplated within the invention.
• the invention further provides use of a yeast strain contemplated within the invention to produce a beer with a low pH and a sour taste.
• the invention further contemplates use of a kit contemplated within the invention to produce a beer with a low pH and a sour taste.
• the method comprises fermenting a wort in the presence of a yeast strain able to produce a yeast-fermented beverage with a low pH and a sour taste without the use of lactic acid producing bacteria.
• the yeast strain is able to produce a yeast-fermented beverage with a pH of about 4.2 to about 3.3 without the use of lactic acid producing bacteria.
• the yeast-fermented beverage is beer.
• the beer is produced without the use of lactic acid producing bacteria.
• the beer is produced without the use of lactic acid.
• the beer has a pH of about 4.2 to about 3.3.
• the yeast strain is able to reduce the pH of wort to about 3.5 in about 5 days without the use of acid producing bacteria.
• the yeast strain belongs to the genus Lachancea.
• the yeast strain is GY7B
• the yeast strain comprises the nucleotide sequence of SEQ ID NO. 8 within the ITS region. In yet other embodiments, the yeast strain comprises the nucleotide sequence of SEQ ID NO. 9 within the actinl gene. In yet other embodiments, the yeast strain comprises the nucleotide sequence of SEQ ID NO. 8 within the ITS region and the nucleotide sequence of SEQ ID NO. 9 within the actinl gene.
• the method produces a yeast-fermented beverage with a pH of about 4.2 to about 3.3.
• the wort is fermented in the absence of any acid producing bacteria.
• the method does not comprise use of lactic acid, or equivalent thereof.
• lactic acid, or equivalent thereof is not added to the wort, before, during, and/or after fermentation.
• the wort is fermented in the absence of any lactic acid producing bacteria. In other embodiments, the wort is fermented in the absence of bacteria belonging to genera Lactobacillus. In yet other embodiments, the wort is fermented in the absence of bacteria belonging to genera Pediococcus. In yet other embodiments, the wort is fermented in the absence of bacteria belonging to genera Lactobacillus and 'or Pediococcus. In yet other embodiments, the wort comprises malt derived from one or more grains selected from the group consisting of barley, wheat, com, rye, rice, oats, sorghum, millet, buckwheat, quinoa, and teff
• the wort is fermented in the presence of at least one additional yeast strain.
• the yeast strain is part of a composition further comprising at least one additional yeast selected from the group consisting of a Saccharomyces yeast, another Lachancea yeast, and a Brettanomyces yeast.
• the yeast strain is part of a composition further comprising at least one Saccharomyces yeast.
• the yeast strain is part of a composition further comprising at least one additional Lachancea yeast.
• the yeast strain is part of a composition further comprising at least one Brettanomyces yeast.
• the composition further comprises at least one additional yeast.
• the at least one additional yeast is selected from the group consisting of a Saccharomyces yeast, another Lachancea yeast, and a Brettanomyces yeast.
• the at least one additional yeast is a Saccharomyces yeast.
• the at least one additional yeast is another Lachancea yeast.
• the at least one additional yeast is a Brettanomyces yeast.
• the yeast strain is able to produce a yeast-fermented beverage with a pH of about 4.2 to about 3.3 without the use of lactic acid producing bacteria. In other embodiments, the yeast strain is able to reduce the pH of word to about 3.5 in about 5 days without the use of acid producing bacteria.
• the yeast strain belongs to the genus Lachancea. In other embodiments, the yeast strain is GY7B (deposited with the ATCC under Accession Number on July 19, 2018). In yet other embodiments, the yeast strain comprises the nucleotide sequence of SEQ ID NO. 8 within the ITS region. In yet other embodiments, the yeast strain comprises the nucleotide sequence of SEQ ID NO. 9 within the actinl gene. In yet other embodiments, the yeast strain comprises the nucleotide sequence of SEQ ID NO. 8 within the ITS region and the nucleotide sequence of SEQ ID NO. 9 within the actinl gene.
• the kit comprises a yeast strain contemplated within the invention and one or more items or ingredients suitable to produce a yeast-fermented beverage.
• the kit comprises yeast strain GY7B (deposited with the
• the kit further comprises instructional materials comprising instructions for producing a yeast- fermented beverage using yeast strain GY7B.
• the kit further comprises one or more items or ingredients to produce a yeast-fermented beverage using GY7B;
• the kit comprises a yeast strain from the genus achancea, wherein the yeast strain comprises the nucleotide sequence of SEQ ID NO. 8 within the ITS region and/or the nucleotide sequence of SEQ ID NO. 9 within the actinl gene.
• the kit comprises instructional materials comprising instructions for producing a yeast-fermented beverage using the yeast strain.
• the kit comprises one or more items or ingredients to produce a yeast-fermented beverage using the yeast strain.
• the one or more items or ingredients comprise prepared wort solution. In other embodiments, the one or more items or ingredients comprise dry malt extract. In yet other embodiments, the one or more items or ingredients comprise one or more grains selected from the group consisting of barley, wheat, com, rye, rice, oats, sorghum, millet, buckwheat, quinoa, and teff In yet other embodiments, the one or more items or ingredients comprise at least one additional yeast strain. In yet other embodiments, the one or more items or ingredients comprise one or more varieties of hops. In yet other embodiments, the one or more items or ingredients comprise conditioned brewing water. In yet other embodiments, the one or more items or ingredients comprise one or more sugar adjuncts.
• the at least one additional yeast strain is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces pastor ianus, Saccharomyces paradoxus, Saccharomyces eubayanus, Saccharomyces ludwigii, Aureobasidium pullulans, Cyberlindnera saturnus, Hansensiaspora uvarum, Hansensiaspora guilliermondii,
• Wickerhamomyces subpelliculosus Wickerhamomyces anomalus, Zygosaccharomyces rouxii, Zygosaccharomyces bailii, Zygosaccharomyces fermentati, Zygosaccharomycesflorentinus, Kluyveromyces lactis, Kluyveromyces marxianus, Lachancea thermotolerans, Brettanomyces bruxellensis, Brettanomyces anomalus, Brettanomyces custersianus, Brettanomyces naardenensis, Brettanomyces nanus, Dekkera bruxellensis, and Dekkera anomala.
• the at least one additional yeast strain is Saccharomyces cerevisiae. In other embodiments, the at least one additional yeast strain is Saccharomyces pastorianus. In yet other embodiments, the at least one additional yeast strain is
• the at least one additional yeast strain is Saccharomyces eubayanus. In yet other embodiments, the at least one additional yeast strain is Saccharomyces ludwigii. In yet other embodiments, the at least one additional yeast strain is Aureobasidium pullulans. In yet other embodiments, the at least one additional yeast strain is Cyberlindnera saturnus. In yet other embodiments, the at least one additional yeast strain is Hansensiaspora uvarum. In yet other embodiments, the at least one additional yeast strain is Hansensiaspora guilliermondii. In yet other embodiments, the at least one additional yeast strain is Hansensiaspora osmophila.
• the at least one additional yeast strain is Hansensiasporavineae. In yet other embodiments, the at least one additional yeast strain is Hansenula anomala. In yet other embodiments, the at least one additional yeast strain is Issatchenkia occidentalis. In yet other embodiments, the at least one additional yeast strain is Issatchenkia orientalis. In yet other embodiments, the at least one additional yeast strain is Pichia kluyveri. In yet other embodiments, the at least one additional yeast strain is Pichia caribbica. In yet other embodiments, the at least one additional yeast strain is Pichia fermentans. In yet other embodiments, the at least one additional yeast strain is Pichia kudriavzevii.
• the at least one additional yeast strain is Pichia Membranifaciens . In yet other embodiments, the at least one additional yeast strain is Rhodotorula mucilaginosa. In yet other embodiments, the at least one additional yeast strain is Torulaspora delbrueckii. In yet other embodiments, the at least one additional yeast strain is Candida colliculosa. In yet other embodiments, the at least one additional yeast strain is Candida shehatae. In yet other embodiments, the at least one additional yeast strain is Candida tropicalis. In yet other embodiments, the at least one additional yeast strain is Candida ethanolica. In yet other embodiments, the at least one additional yeast strain is Candida krusei.
• the at least one additional yeast strain is Candida magnolia. In yet other embodiments, the at least one additional yeast strain is Candida milleri. In yet other embodiments, the at least one additional yeast strain is Clavispora lusitaniae. In yet other embodiments, the at least one additional yeast strain is Wickerhamomyces subpelliculosus. In yet other embodiments, the at least one additional yeast strain is Wickerhamomyces anomalus. In yet other embodiments, the at least one additional yeast strain is Zygosaccharomyces rouxii. In yet other
• the at least one additional yeast strain is Zygosaccharomyces bailii. In yet other embodiments, the at least one additional yeast strain is Zygosaccharomyces fermentati. In yet other embodiments, the at least one additional yeast strain is
• the at least one additional yeast strain is Kluyveromyces lactis. In yet other embodiments, the at least one additional yeast strain is Kluyveromyces marxianus. In yet other embodiments, the at least one additional yeast strain is Lachancea thermotolerans . In yet other embodiments, the at least one additional yeast strain is Brettanomyces bruxellensis . In yet other embodiments, the at least one additional yeast strain is Brettanomyces anomalus. In yet other embodiments, the at least one additional yeast strain is Brettanomyces custersianus. In yet other embodiments, the at least one additional yeast strain is Brettanomyces naardenensis .
• the at least one additional yeast strain is Brettanomyces nanus. In yet other embodiments, the at least one additional yeast strain is Dekkera bruxellensis . In yet other embodiments, the at least one additional yeast strain is Dekkera anomala.
• the wort comprises hops.
• the hops comprises Ahtanum.
• the hops comprises Amarillo.
• the hops comprises Apollo.
• the hops comprises Cascade.
• the hops comprises Centennial. In yet other
• the hops comprises Chinook. In yet other embodiments, the hops comprises Citra. In yet other embodiments, the hops comprises Cluster. In yet other embodiments, the hops comprises Columbus. In yet other embodiments, the hops comprises Crystal. In yet other embodiments, the hops comprises Eroica. In yet other embodiments, the hops comprises Galena. In yet other embodiments, the hops comprises Glacier. In yet other embodiments, the hops comprises Greenburg. In yet other embodiments, the hops comprises Horizon. In yet other embodiments, the hops comprises Liberty. In yet other embodiments, the hops comprises Millenium. In yet other embodiments, the hops comprises Mount Hood. In yet other embodiments, the hops comprises Mount Rainier. In yet other embodiments, the hops comprises Newport.
• the hops comprises Nugget. In yet other embodiments, the hops comprises Palisade. In yet other embodiments, the hops comprises Santiam. In yet other embodiments, the hops comprises Simcoe. In yet other embodiments, the hops comprises Sterling. In yet other embodiments, the hops comprises Summit. In yet other embodiments, the hops comprises Tomahawk. In yet other
• the hops comprises Ultra. In yet other embodiments, the hops comprises Vanguard. In yet other embodiments, the hops comprises Warrior. In yet other
• the hops comprises Willamette. In yet other embodiments, the hops comprises Zeus. In yet other embodiments, the hops comprises Admiral. In yet other embodiments, the hops comprises Brewer's Gold. In yet other embodiments, the hops comprises Bullion. In yet other embodiments, the hops comprises Challenger. In yet other embodiments, the hops comprises First Gold. In yet other embodiments, the hops comprises Fuggles. In yet other embodiments, the hops comprises Goldings. In yet other embodiments, the hops comprises Herald. In yet other embodiments, the hops comprises Northdown. In yet other
• the hops comprises Northern Brewer. In yet other embodiments, the hops comprises Phoenix. In yet other embodiments, the hops comprises Pilot. In yet other embodiments, the hops comprises Pioneer. In yet other embodiments, the hops comprises Progress. In yet other embodiments, the hops comprises Target. In yet other embodiments, the hops comprises Whitbread Golding Variety (WGV). In yet other embodiments, the hops comprises Hallertau. In yet other embodiments, the hops comprises Hersbrucker. In yet other embodiments, the hops comprises Saaz. In yet other embodiments, the hops comprises Tettnang. In yet other embodiments, the hops comprises Spalt. In yet other embodiments, the hops comprises Feux-Coeur Francais.
• the hops comprises Galaxy. In yet other embodiments, the hops comprises Green Bullet. In yet other embodiments, the hops comprises Motueka. In yet other embodiments, the hops comprises Nelson Sauvin. In yet other embodiments, the hops comprises Pacific Gem. In yet other embodiments, the hops comprises Pacific Jade. In yet other embodiments, the hops comprises Pacifica. In yet other embodiments, the hops comprises Pride of Ringwood. In yet other embodiments, the hops comprises Riwaka. In yet other embodiments, the hops comprises Southern Cross. In yet other embodiments, the hops comprises Lublin. In yet other embodiments, the hops comprises Magnum. In yet other embodiments, the hops comprises Perle.
• the hops comprises Polnischer Lublin. In yet other embodiments, the hops comprises Saphir. In yet other embodiments, the hops comprises Satus. In yet other embodiments, the hops comprises Select. In yet other embodiments, the hops comprises Strisselspalt. In yet other embodiments, the hops comprises Styrian Goldings. In yet other embodiments, the hops comprises Tardif de Bourgogne. In yet other embodiments, the hops comprises tradition. In yet other embodiments, the hops comprises Bravo. In yet other embodiments, the hops comprises Calypso. In yet other embodiments, the hops comprises Chelan. In yet other embodiments, the hops comprises Comet. In yet other embodiments, the hops comprises El Dorado.
• the hops comprises San Juan Ruby Red. In yet other embodiments, the hops comprises Satus. In yet other embodiments, the hops comprises Sonnet Golding. In yet other embodiments, the hops comprises Super Galena. In yet other embodiments, the hops comprises Tillicum. In yet other embodiments, the hops comprises Branding Cross. In yet other embodiments, the hops comprises Pilgrim. Hallertauer Herkules. In yet other embodiments, the hops comprises Hallertauer Magnum. In yet other embodiments, the hops comprises Hallertauer Taurus. In yet other embodiments, the hops comprises Merkur. In yet other embodiments, the hops comprises Opal. In yet other embodiments, the hops comprises Smaragd.
• the hops comprises Halleratau Aroma. In yet other embodiments, the hops comprises Kohatu. In yet other embodiments, the hops comprises Rakau. In yet other embodiments, the hops comprises Stella. In yet other embodiments, the hops comprises Sticklebract. In yet other embodiments, the hops comprises Summer Saaz. In yet other embodiments, the hops comprises Super Alpha. In yet other embodiments, the hops comprises Super Pride. In yet other embodiments, the hops comprises Topaz. In yet other embodiments, the hops comprises Wai-iti. In yet other embodiments, the hops comprises Bor. In yet other embodiments, the hops comprises Junga. In yet other embodiments, the hops comprises Marynka.
• the hops comprises Premiant. In yet other embodiments, the hops comprises Sladek. In yet other embodiments, the hops comprises Styrian Atlas. In yet other embodiments, the hops comprises Styrian Aurora. In yet other embodiments, the hops comprises Styrian Bobek. In yet other embodiments, the hops comprises Styrian Celeia. In yet other embodiments, the hops comprises Sybilla. In yet other embodiments, the hops comprises Sorachi Ace.
• FIGs. 1A-1C are a non-limiting illustrative diagram outlining the methods used to select, isolate and characterize yeast strains of potential interest.
• Yeast strains of potential interest can be selected at step 102 where collected yeast strains are cultured with wort containing antibiotics and assessed for CO2 production, wort density reduction, and tolerance for high dissolved sugar concentrations.
• FIGs. 2A-2B are graphs reporting pH (FIG. 2A) and apparent extract (FIG. 2B) of fermentation trials of 10% light dry malt extract (DME) unhoped wort using Saccharomyces cerevisiae ⁇ Belle Hong strain; Lallemand), a newly discovered yeast strain GY7B, and a mixture of both strains.
• the wort was incubated at 20°C and inoculated with lxl 0 6 yeast cells per milliliter per degree Plato.
• the GY7B strain demonstrated greater acidification and a greater longevity than the Saccharomyces cerevisiae or the mixed strain samples. As demonstrated in FIG.
• GY7B does not ferment as quickly as the Saccharomyces cerevisiae strain used in this trial. Without being limited to any one theory, it is possible that the lack of acidification observed in co-fermentation between GY7B and Saccharomyces cerevisiae is due to the faster fermentation abilities of traditional ale yeast strains that outcompetes GY7B's ability to create lactic acid.
• FIG 2C is a graph reporting pH and apparent extract of a brewer's wort using Saccharomyces cerevisiae (American Ale II strain; Wyeast 1272), GY7B, and Lachancea thermotolerans; type strain NRRL Y-8284.
• Saccharomyces cerevisiae American Ale II strain; Wyeast 1272
• GY7B fermented faster than L. thermotolerans but slower than S. cerevisiae.
• GY7B exhibited rapid souring during fermentation, reaching pH 3.5 in 4 days.
• FIGs. 4A-4B are graphs showing the effect of pH on growth rates for GY7B (FIG. 4A) and standard Lachancea thermotolerans; NRRL Y-8284 (FIG. 4B) yeast over time. GY7B exhibited greater tolerance for low pH than L. thermotolerans.
• FIGs. 5A-5B are graphs showing effect of ethanol concentration on growth rates for GY7B (FIG. 5A) and Lachancea thermotolerans NRRL Y8284 (FIG. 5B) yeast over time. GY7B exhibited greater tolerance for high ethanol concentrations than L. thermotolerans.
• FIG. 6 is an image comparing the fiocculation characteristics of GY7B and standard Lachancea thermotolerans; NRLL Y-8284. GY7B is highly flocculant while L.
• thermotolerans is not.
• the present invention relates to the unexpected discovery of a new strain of yeast, dubbed GY7B, which is related to, but genetically and phenotypically distinct from,
• the invention provides methods of brewing a yeast-fermented beverage, such as but not limited to sour beer, using GY7B, wherein the methods do not require use of lactic acid and/or LAB.
• the methods of the invention can produce a wort with a pH of about 3.5 after fermenting for 5 days in the presence of GY7B, while methods known in the art using bacteria can take months to reach the same pH.
• the methods of the invention are performed without addition of lactic acid, or equivalents thereof, to the compositions contemplated within the invention.
• lactic acid, or any equivalents thereof is not added during fermentation.
• lactic acid, or any equivalents thereof is not added after fermentation.
• the yeast contemplated within the invention is typically not of the Lachancea thermotolerans species. Definitions
• the articles “a” and “an” refer to one or to more than one (i.e. , to at least one) of the grammatical object of the article.
• an element means one element or more than one element.
• the term "about” is understood by persons of ordinary skill in the art and varies to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of ⁇ 20% or ⁇ 10%, more preferably ⁇ 5%, even more preferably ⁇ 1%, and still more preferably ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
• the term "GY7B” may be referred to as the yeast deposited with the ATCC under Accession Number on July 19, 2018.
• the GY7B yeast strain is characterized for having the nucleotide sequence of SEQ ID NO. 8 within the ITS region.
• the GY7B yeast strain is characterized for having the nucleotide sequence of SEQ ID NO. 9 within the actinl gene.
• the GY7B yeast strain is characterized for having the nucleotide sequence of SEQ ID NO. 8 within the ITS region and the nucleotide sequence of SEQ ID NO. 9 within the actinl gene.
• “Instructional material,” as that term is used herein, includes a publication, a recording, a diagram, or any other medium of expression that can be used to communicate the usefulness of the composition and/or compound of the invention in a kit.
• the instructional material of the kit may, for example, be affixed to a container that contains the compound and/or composition of the invention or be shipped together with a container that contains the compound and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively. Delivery of the instructional material may be, for example, by physical delivery of the publication or other medium of expression
• communicating the usefulness of the kit may alternatively be achieved by electronic transmission, for example by means of a computer, such as by electronic mail, or download from a website.
• low pH in a fermented beverage indicates that the pH of the beverage is higher than or equal to about 2.5, and lower than or equal to about 4.5.
• the upper limit of low pH can be lower than or equal to about 4.5, lower than or equal to about 4.4, lower than or equal to about 4.3, lower than or equal to about 4.2, lower than or equal to about 4.1, and/or lower than or equal to about 4.0.
• the lower limit of the pH can be set as higher than or equal to about 2.5, higher than or equal to about 2.6, higher than or equal to about 2.7, higher than or equal to about 2.8, higher than or equal to about 2.9, higher than or equal to about 3.0, higher than or equal to about 3.1, higher than or equal to about 3.2, higher than or equal to about 3.3, higher than or equal to about 3.4, and/or higher than or equal to about 3.5. Any numerical ranges having the upper limits and the lower limits as shown elsewhere herein can be adopted.
• the pH of the beverage can be in ranges of about 2.7 or higher, and about 4.5 or lower; about 2.7 or higher, and about 4.2 or lower; about 2.7 or higher, and about 4.0 or lower; about 3.0 or higher, and about 4.5 or lower; about 3.0 or higher, and about 4.2 or lower; about 3.1 or higher, and about 4.2 or lower; about 3.2 or higher, and about 4.2 or lower; about 3.3 or higher, and about 4.2 or lower; about 3.0 or higher, and about 4.0 or lower; about 3.5 or higher, and about 4.5 or lower; about 3.5 or higher, and about 4.2 or lower; and/or about 3.5 or higher, and about 4.0 or lower.
• the pH is about 2.5.
• the pH is about 2.6. In yet other embodiments, the pH is about 2.7. In yet other embodiments, the pH is about 2.8. In yet other embodiments, the pH is about 2.9. In yet other embodiments, the pH is about 3.0. In yet other embodiments, the pH is about 3.1. In yet other embodiments, the pH is about 3.2. In yet other embodiments, the pH is about 3.3. In yet other embodiments, the pH is about 3.4. In yet other embodiments, the pH is about 3.5. In yet other embodiments, the pH is about 3.6. In yet other embodiments, the pH is about 3.7. In yet other embodiments, the pH is about 3.8. In yet other embodiments, the pH is about 3.9. In yet other embodiments, the pH is about 4.0. In yet other embodiments, the pH is about 4.1. In yet other embodiments, the pH is about 4.2. In yet other embodiments, the pH is about 4.3. In yet other
• the pH is about 4.4. In yet other embodiments, the pH is about 4.5.
• the term "mash” is understood to mean a mix of milled grains and water used in brewing and distilling processes to produce wort. Typically malted grains are heated in order to breakdown the starch in the grains into simple sugars, which can then be fermented in order to produce alcohol.
• the "Plato scale” or “Plato gravity scale” refers to the empirically derived hydrometer scale used to measure the density of beer wort in terms of percentage of extract by weight.
• the terms “degrees Plato” or “° Plato” or “° P” are units of measurement on the Plato scale.
• the term "sour" in a fermented beverage refers to an acid, bitter, and/or sharp taste in the palate caused by the beverage.
• the sour taste is associated with the low pH of the fermented beverage.
• wort is understood to mean the liquid extracted from the mashing process during a brewing or distilling process.
• Wort contains the sugars extracted from the malted grains which will be fermented by the brewing yeast in order to produce alcohol.
• range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range and, when appropriate, partial integers of the numerical values within ranges.
• range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
• BLAST Basic local alignment search tool
• DME dry malt extract
• IBU International Bittering Units
• LAB lactic acid bacteria
• nt nucleotide
• OD Optical Density
• PCR polymerase chain reaction
• P/S Penicillin/ Streptomycin
• YPD yeast extract peptone dextrose.
• the invention provides a yeast strain able to produce a yeast-fermented beverage with a low pH and a sour taste without the use of lactic acid producing bacteria.
• the yeast strain is able to produce a yeast-fermented beverage with a pH of about 4.2 to about 3.3 without the use of lactic acid producing bacteria.
• the yeast strain is able to reduce the pH of word to about 3.5 in about 5 days without the use of acid producing bacteria.
• the yeast strain belongs to the genus Lachancea.
• the yeast strain is GY7B (deposited with the ATCC under Accession Number on July 19, 2018).
• the yeast strain comprises the nucleotide sequence of SEQ ID NO.
• the yeast strain comprises the nucleotide sequence of SEQ ID NO. 9 within the actinl gene. In yet other embodiments, the yeast strain comprises the nucleotide sequence of SEQ ID NO. 8 within the ITS region and the nucleotide sequence of SEQ ID NO. 9 within the actinl gene.
• the yeast strain of the invention is part of a composition.
• the composition is compatible with fermentation processes.
• the composition further comprises at least one additional yeast selected from the group consisting of Saccharomyces, Lachancea, and Brettanomyces.
• the composition further comprises a Saccharomyces yeast.
• the composition further comprises a distinct Lachancea yeast.
• the composition further comprises a Brettanomyces yeast.
• the present invention provides a method of producing a yeast fermented beverage with a low pH and sour taste without the use of lactic acid producing bacteria.
• the invention provides a method of producing a yeast- fermented beverage.
• the method comprises fermenting a wort in the presence of a yeast strain able to produce a yeast-fermented beverage with a low pH and a sour taste without the use of lactic acid producing bacteria.
• the yeast-fermented beverage is beer.
• the method does not comprise use of lactic acid, or any equivalent thereof.
• lactic acid, or any equivalent thereof is not added to the wort, before, during, or after fermentation.
• the yeast strain is able to produce a yeast-fermented beverage with a pH of about 4.2 to about 3.3 without the use of lactic acid producing bacteria. In other embodiments, the yeast strain is able to reduce the pH of wort to about 3.5 in about 5 days without the use of acid producing bacteria. In yet other embodiments, the yeast strain belongs to the genus Lachancea. In yet other embodiments, the yeast strain is GY7B (deposited with the ATCC under Accession Number on July 19, 2018). In yet other embodiments, the yeast strain comprises the nucleotide sequence of SEQ ID NO. 8 within the ITS region. In yet other embodiments, the yeast strain comprises the nucleotide sequence of SEQ ID NO. 9 within the actinl gene. In yet other embodiments, the yeast strain comprises the nucleotide sequence of SEQ ID NO. 8 within the ITS region and the nucleotide sequence of SEQ ID NO. 9 within the actinl gene.
• the method produces a yeast-fermented beverage with a pH of about 4.2 to about 3.3.
• the wort is fermented in the absence of any acid producing bacteria.
• the wort is fermented in the absence of any lactic acid producing bacteria.
• the wort is fermented in the absence of bacteria belonging to genera Lactobacillus or Pediococcus.
• the wort comprises malt derived from one or more grains selected from the group consisting of barley, wheat, corn, rye, rice, oats, sorghum, millet, buckwheat, quinoa, and teff.
• the wort further comprises hops.
• the wort is fermented in the presence of at least one additional yeast strain selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces paradoxus, Saccharomyces eubayanus, Saccharomyces ludwigii,
• the hops is at least one selected from the group consisting of
• the invention further provides use of a yeast strain contemplated herein, and/or a kit contemplated herein, to produce a beer with a low pH and a sour taste.
• a yeast strain contemplated herein and/or a kit contemplated herein, to produce a beer with a low pH and a sour taste.
• the beer is produced without the use of lactic acid producing bacteria. In other embodiments, the beer is produced without the use of lactic acid. In yet other embodiments, the beer has a pH of about 4.2 to about 3.3.
• the method comprises fermenting wort in the presence of GY7B yeast. In certain embodiments, the method produces the sour tasting yeast fermented beverage faster than known methods that use lactic acid producing bacteria.
• the yeast fermented beverage is a beer.
• beer is a sour beer similar in style to a beer selected from the group consisting of Iambic, geuze (gueuze),Mon, farmhouse, framboise, kriek, Principle weisse, Flanders red ale, oud bruin, gose and American wild ale.
• the GY7B yeast reduces the pH of wort to about 3.5 in about
• the GY7B yeast is more tolerant to low pH than common brewers yeasts known in the art, such as but not limited to Saccharomyces cerevisiae and Lachancea thermotolerans .
• the GY7B yeast thrives in environments as low as pH 3.
• the GY7B yeast produces a beer with a pH of about 4.2 to about 3.3.
• the GY7B yeast tolerates alcohol concentrations up to about 10% vol/vol alcohol.
• Malt can be prepared from dried, germinated cereal grains (mainly barley or wheat) and grounded into a grist that may contain unmalted adjuncts.
• the grist can be mashed (mixed with water and steeped) to allow enzymes in the malt to convert the starch into sugars.
• the grain particles and adjuncts can be separated from the liquid wort in a process called lautering.
• the malt making and mashing steps can be skipped by adding water to malt extract.
• the wort After addition of hops and/or other ingredients such as herbs and sugars, the wort can be boiled (hops may also be added after boiling), cooled and aerated.
• the wort can then be moved to a fermentation tank and fermented by the addition of a brewer's yeast.
• the primary fermentation lasting typically 5 to 10 days, may be followed by a secondary fermentation step using a further brewer's yeast.
• After fermentation the fresh beer or "green” beer can be conditioned, optionally filtrated and carbonated.
• Hops are added to the wort to balance the sweetness of the malt with bitterness and impart onto the beer desirable flavors and aromas.
• the yeast fermented beverage is produced by fermenting wort derived from a mash comprising one or more grains selected from the group consisting of barley, wheat, corn, rye, rice, oats, sorghum, millet, buckwheat, quinoa, teff, dry malt extract, and liquid malt extract.
• the yeast fermented beverage may contain additional fermentable sugar as provided by adjuncts such as, but not limited to purified sugars or syrups.
• the mash comprises malted grains.
• the method further comprises the addition of flowers of the hop plant ("hops"), Humulus lupulus, to the wort.
• the hops are added to the wort before addition of the GY7B yeast.
• the hops are added to the fermenting beer after addition of the GY7B yeast.
• the method comprises fermenting wort in the presence of GY7B yeast alone or in the presence of at least one additional yeast strain selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces paradoxus, Saccharomyces eubayanus, Saccharomyces ludwigii, Aureobasidium pullulans, Cyberlindnera saturnus Hansensiaspora uvarum Hansensiaspora guilliermondii,
• Wickerhamomyces subpelliculosus Wickerhamomyces anomalus, Zygosaccharomyces rouxii, Zygosaccharomyces bailii, Zygosaccharomyces fermentati, Zygosaccharomycesflorentinus, Kluyveromyces lactis, Kluyveromyces marxianus, Lachancea thermotolerans, Brettanomyces bruxellensis, Brettanomyces anomalus, Brettanomyces custersianus, Brettanomyces naardenensis, Brettanomyces nanus, Dekkera bruxellensis and Dekkera anomala.
• the starting gravity of the wort is from about 6°P to about
• the starting gravity of the wort is about 10°P to about 16°P.
• the concentration of GY7B in the wort is about lxl 0 6 cells/mL to about 2xl0 7 cells/ml.
• the wort is fermented at a temperature of about 9°C to about 30°C. In other embodiments, the wort is fermented at about 20°C.
• the method requires minimal, or no, filtering.
• GY7B demonstrates high fiocculation and rapidly settles to the bottom of the fermenting vessel, easing separation.
• the method facilitates fiocculation of additional yeast strains during co-fermentation.
• the method further comprises pasteurization.
• the fermented wort is pasteurized at 15-30 Pasteurization Units.
• the GY7B is added to the fermenter. In other embodiments, GY7B is added to the mash. In other embodiments, GY7B is added to a secondary fermentation or maturation vessel such as a fermenter, barrel, foeder, bright tank, keg, cask, can, or bottle.
• the invention provides a kit comprising at least one yeast strain contemplated herein and one or more items or ingredients suitable to produce a yeast-fermented beverage.
• the invention further provides a kit comprising yeast strain GY7B (deposited with the ATCC under Accession Number on July 19, 2018), and one or more items or ingredients suitable to produce a yeast-fermented beverage.
• the invention further provides a kit comprising a yeast strain from the genus Lachancea, wherein the yeast strain comprises the nucleotide sequence of SEQ ID NO. 8 within the ITS region and/or the nucleotide sequence of SEQ ID NO. 9 within the actinl gene, and one or more items or ingredients suitable to produce a yeast-fermented beverage.
• the kit further comprises instructional materials comprising instructions for producing a yeast-fermented beverage using the yeast strain contemplated.
• the yeast-fermented beverage is beer.
• the one or more items or ingredients comprise at least one item selected from the group consisting of: prepared wort solution; dry malt extract; one or more grains selected from the group consisting of barley, wheat, com, rye, rice, oats, sorghum, millet, buckwheat, quinoa, and teff; at least one additional yeast strain selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces paradoxus, Saccharomyces eubayanus, Saccharomyces ludwigii,
• kits comprising GY7B and items and ingredients necessary to brew beer.
• the kit comprises packaged GY7B. In other embodiments, the kit comprises dried GY7B in the form of a powder. In other embodiments, the kit comprises GY7B in a vacuum sealed container. In other embodiments, the kit comprises GY7B from a fresh propagation or a post-fermentation slurry in a vented container.
• the kit further comprises one or more items necessary for performing the methods of the invention selected from the group consisting of one or more vessels adapted and configured for wort fermentation, one or more thermometers, one or more hydrometers, one or more vessels adapted and configured for wort boiling, one or more vessels adapted and configured for pasteurization, one or more vessels adapted and configured for beer storage, one more vessels adapted and configured for aging of beer.
• the kit further comprises one or more items selected from the group consisting of compressed CO2 tanks, compressed N 2 tanks, gas regulators, tubing, and pressure gauges.
• the kit further comprises at least one additional ingredient necessary for performing the methods of the invention selected from the group consisting of prepared wort solution, dry or liquid malt extract, one or more grains selected from the group consisting of barley, wheat, corn, rye, rice, oats, sorghum, millet, buckwheat, quinoa, and teff, at least one additional yeast strain selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces paradoxus, Saccharomyces eubayanus, Saccharomyces ludwigii, Aureobasidium pullulans, Cyberlindnera saturnus Hansensiaspora uvarum Hansensiaspora guilliermondii, Hansensiaspora osmophila, Hansensiasporavineae, Hansenula anomala, Issatchenkia occidentalis, Issatchenki
• Brettanomyces anomalus Brettanomyces custersianus, Brettanomyces naardenensis, Brettanomyces nanus, Dekkera bruxellensis, and Dekkera anomala, one or more varieties of hops, conditioned brewing water, and one or more sugar adjuncts.
• the kit further comprises instructional materials containing instructions for performing the methods of the invention.
• the instructional materials provide information pertaining to brewing beer with GY7B using the items and ingredients of the kit of the invention.
• An exemplary yeast strain of the invention is the GY7B yeast strain. Under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure, deposit of the yeast strain was made with the American Type Culture Collection (ATCC) of Rockville, Md., USA.
• ATCC American Type Culture Collection
• the deposited material will be maintained with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposited material, and in any case, for a period of at least thirty (30) years after the date of the deposit or for the enforceable life of the patent, whichever period is longer.
• Applicants' assignee acknowledges its duty to replace the deposit should the depository be unable to furnish a sample when requested due to the condition of the deposit.
• range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
• Samples for wild yeast isolation were collected using aseptic technique in the field. Samples were then inoculated directly in a 10% DME (BREISS®) solution containing
• Penicillin/Streptomycin (GIBCO®). After two weeks, samples were plated on YPD containing P/S and cultured at 25°C for 48 hours. Individual colonies were selected for further propagation on YPD P/S plates under the same culture conditions. Purity of colonies was validated by uniformity in morphology, at which point colonies were maintained on YPD plates without antibiotics. Glycerol stocks were created by picking a colony and culturing in YPD broth at 25°C, 180rpm for 48 hours. Then cultures were then incubated at 4°C for another 48 hours. Finally, the yeast were resuspended in fresh YPD with 15% glycerol and frozen in cryovials at -80°C. All experiments were run with yeast freshly propagated from glycerol stocks.
• Test fermentations were either performed in 10% DME, unhoped media, referred to as laboratory wort or in wort prepared in the University of the Sciences pilot brewery.
• 10% DME laboratory wort lOOg of DME was dissolved in 1L of distilled water, boiled for 15 minutes, and cooled to room temperature.
• 10°P wort was prepared in the USciences pilot brewery using 100% 2-row pale malt (BREISS®). It was milled and mashed on a SABCOTM BrewMagic using distilled water at 65 °C for 60min. After vorlauf, continuous fly sparging was performed with 75°C distilled water acidified to pH6.0 with food-grade lactic acid (SPECTRUM® chemicals). The wort was boiled for 60 minutes, with a 60 minute bittering addition of 007: The Golden Hop (Yakima Valley Hops) to yield 10IBU of bitterness.
• Whirlfloc (LD CARLSONTM) was added with 15min remaining in the boil as per the manufacturer's instructions.
• the yeast supplement Servomyces (WHITE LABS) was added with lOmin remaining in the boil as per the manufacturer's instructions. After the boil, the wort was brought into a whirlpool and allowed to settle for 15min before chilling to room temperature with the SABCO Chill Wizard. All wort was bottled, autoclaved and stored at 4°C before use.
• YPD agar 2% glucose, 2% peptone, 1% yeast extract, and 1.5% agar was supplemented with a 1 : 100 dilution of P/S as needed. All reagents were from Research Products International.
• yeast identification fresh yeast cultures grown at 25-30°C for ⁇ 48 hours on YPD were used. Crude DNA extraction was performed by selecting a small yeast colony and transferring to 0.2% SDS followed by incubation at 90°C for 4 minutes. The lysed yeast were then diluted 1: 10 in nuclease-free water. This dilution was further diluted 1:40 into the PCR reaction. PCR was performed with Phusion High-fidelity PCR kit (NEW ENGLAND BIOLABS®) as per the manufacturer's instructions on an EPPENDORF® Mastercycler. The D1/D2 domain of the 26S rDNA region was amplified using the primers NL1 (SEQ ID NO. 1: 5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL4 (SEQ ID NO. 2:
• ITS1 SEQ ID NO. 3:
• PCR reactions were resolved on a 1% agarose gel at 120volts (BIO-RAD®) and visualized with ethidium bromide under UV light. Bands were excised and purified using the GeneJet Gel Extraction kit (THERMO FISHER SCIENTIFIC®) according to manufacturer's instructions.
• PCR DNA was prepared for Sanger sequencing by Genewiz according to their instructions. Each PCR product was sequenced with both the forward and the reverse primers used in the original PCR amplification. Returned sequences were validated for accuracy by aligning the forward and the reverse sequences with A Plasmid Editor (M.
• NCBI Nucleotide BLAST
• Example 1 Methods of Collecting and Testing Wild Yeast strains
• samples were collected from Woodlands Registry (4000 Woodland Ave, Philadelphia, PA 19104) from local flowering trees, as well as a local bee hive by swabbing (101) and inoculating in sterile wort (102).
• the samples were incubated without shaking at 25°C for about 1 week. Sediment was gently re-suspended and plated onto YPD plates (103). Once visible colonies were present after 48-72hr growth at 25°C, morphologically distinct colonies were sub-cultured to purity (104).
• the purified yeast strains were subject to PCR of the D1/D2 region to amplify the genetic material for analysis (105).
• the extracted DNA was separated through gel electrophoresis (106), sequenced (107), and analyzed using the Basic Local Alignment Search Tool (BLAST) to find genetic similarities (108).
• Table 1 A lists all strains isolated and their source.
• Table IB lists the similarity between the yeast isolate's D1/D2 sequence and the top BLAST hit.
• test ferments containing laboratory wort were inoculated with the isolated yeast and observed for a period of 7 days. Of the samples collected GY7B was the most robust fermenter as demonstrated by visible CO2 evolution in the sample tube.
• GY7B was isolated from a Cornecaea cornus (Dogwood tree) bud.
• BLAST analysis of the D1/D2 sequence returned a 100% match with 0 nt differences to Lachancea thermotolerans , indicating it is a wild strain related to L. thermotolerans .
• GY4.1 553 Pichia kudriavzevii 100 0
• GY7B was tested for wort fermentation under controlled conditions.
• Laboratory wort was inoculated with lxlO 6 yeast cells/ml/°P of Saccharomyces cerevisiae (strain Belle VMware from Lallemand), GY7B, or both strains each at lxl 0 6 yeast cells/ml/°P.
• the ferments were incubated at 20°C for 5 days.
• the pH and apparent extract of the ferments were tracked daily using a pH meter and a densitometer, respectively.
• GY7B reduced the pH of the wort to about 3.5 after 5 days, while both the S. cerevisiae and mixed strain samples reduced the pH of the wort to only about 4.25 after 5 days (FIG. 2A).
• GY7B was found to be a slower fermenting yeast than S. cerevisiae, as both the S. cerevisiae and mixed strain samples demonstrated a faster decrease in °P over 5 days than the GY7B sample (FIG. 2B). Without being limited to any particular theory, it is suspected that the lack of acidification observed in co-fermentation between GY7B and Saccharomyces cerevisiae is due to the faster fermentation abilities of S. cerevisiae which outcompetes GY7B's ability to create lactic acid. Optimization of cell inoculation rates during co- fermentation allows the timely production of sour beer via GY7B with the benefits of fermentation with a traditional ale strain.
• thermotolarans type strain NRRL Y-8284
• a traditional Saccharomyces cerevisiae ale strain American Ale II, Wyeast 1272. Brewer's wort with an original gravity of 1.040 and a pH 5.25 was created at the University of the Sciences pilot brewery. Triplicate fermentations of 400mL in Erlenmeyer flasks sealed with a fermentation airlock were inoculated with lxl 0 6 yeast cells / mL / °P. Apparent extract and pH were measured daily at the same time with the averages reported in FIG. 2C. The standard deviation is reported as error bars. As expected, S. cerevisiae rapidly ferments the wort to a final gravity of 1.003 in 4 days. The apparent extract of the wort fermented by L. thermotolerans is only 1.027 after 9 days of fermentation. GY7B ferments slowly but gradually reaching 1.011 apparent attenuation after 9 days.
• the pH and ethanol tolerance of GY7B and L. thermotolerans were evaluated by measuring growth at variable pH (FIG. 4A-5B) or ethanol percentages (v/v) (FIG. 5A-5B).
• Freshly cultured yeast were propagated in 5mL of YPD, shaken at 180rpm at 30° overnight.
• a spectrophotometer was used to measure the OD 60 o and the culture was diluted with fresh YPD to reach an OD 6 oo of 0.1.
• each sample was spiked with 100% Ethanol to reach a final concentration of 0%, 4%, 6%, 8%, or 10% (v/v).
• YPD YPD was acidified with Hydrochloric acid to cover a pH range from 3-7 which was then used to re-suspend yeast after centrifugation.
• 200 ⁇ of OD 6 oo 0.1 culture was placed in a well of a clear, round-bottom 96-well plate (Coming).
• a TEC AN fluorimeter was used to read the OD 6 oo every 5 minutes. Throughout the experiment the fluorimeter was held at 30° with 4 minutes of orbital shaking at 120rpm and 1 minute of linear shaking at 270rpm in between each read. All samples were done in triplicate with the average values reported. The standard deviation is displayed as error bars.
• GY7B is more acid tolerant and ethanol tolerant than L. thermotolerans . Both GY7B and L. thermotolerans can proliferate in up to 10% ethanol (v/v).
• GY7B was performed using YT Microplates (BIOLOG®). Freshly propagated yeast colonies on YPD agar were selected 48-72 hours after growth at 25-30°C. A single colony was inoculated into 15mL of sterile water and was adjusted as needed to yield a % transmittance of 47% ⁇ 2% as determined by spectrophotometer. ⁇ was then placed in each of the 96 wells of the YT Microplate. The plate was incubated at 27°C in a humidified chamber for about 3 days and the results were interpreted by visual inspection. To validate the results, each experiment was repeated twice by two independent analysts. Shown in Table 3 are the concensus results.
• Table 3 is arranged according to the design of the YT Microplate. The first three rows, shaded in gray are oxidation tests that utilize a patented color change in response to yeast cell metabolism. The remaining wells are growth assimilation tests in which turbidity of the well is recorded. Table 3 demonstrates the metabolic and assimilation differences between GY7B and L. thermotolerans.
• thermotolerans exhibits low flocculation as demonstrated by its dispersed pellet and cloudy beer.
• GY7B is highly flocculant as demonstrated by its remaining pellet and clear beer (FIG. 6).
• the high flocculation ability of GY7B will greatly assist yeast removal after fermentation of beer, enabling more effective centrifugation, filtration, pasteurization or any other stabilizing / clarifying processes post-fermentation.

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